Field of the Invention
The present invention relates to methods and compositions for inhibiting botulinum neurotoxins. Specifically, the invention provides quinolinol-based compositions and methods of using quinolinol-based compositions to inhibit the toxic effects of serotype A Clostridium botulinum neurotoxins.
Description of the Related Art
Botulinum neurotoxins (BoNTs), produced by the anaerobic, Gram-positive bacteria Clostridium botulinum, C. baratii, and C. butyricum, consist of seven immunologically distinct serotypes (types A-G). Botulinum neurotoxins are synthesized as ˜150-kDa single-chain protoxins that are post-translationally processed by proteolytic cleavage to form a disulfide-linked dimer composed of a 100-kDa heavy chain (HC) and a 50-kDa light chain (LC) (27, 31, 35, 36). The HC comprises a 50-kDa C-terminal domain (Hc) that participates in the binding of toxin to productive ectoacceptors on the cell surface of peripheral cholinergic nerve cells (3). Toxin is taken up into the cell by receptor-mediated endocytosis (4) and the 50-kDa N-terminal domain (Hn) of the HC facilitates the translocation of the LC across an endosomal membrane into the cytosol of the nerve cell (30). The LC is a zinc-dependent endopeptidase that cleaves and inactivates SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: SNAP-25, VAMP/synaptobrevin, and syntaxin (31, 36). SNARE proteins are essential for exocytosis of neurotransmitter, and cleavage of SNARE protein(s) by BoNT inhibits the release of acetylcholine from synaptic terminals leading to neuromuscular paralysis or botulism (31, 35).
Worldwide, about 1000 cases of human BoNT poisoning, predominantly caused by serotypes A and B, are reported yearly (17). In spite of advances in food production and storage/handling processes, cases of food-borne botulism persist, including a massive outbreak in Thailand (26) and the recent US botulism scare associated with canned chili and other products (2, 28).
Therapy for botulism consists of immunological intervention to neutralize and clear toxin from the circulation, and supportive care, which may include intubation and ventilatory assistance. However, while antibody therapy can be very effective, it has several limitations, including limited availability, lot-to-lot potency variability and short window of application. Clearly, there is a need for improved therapies and compounds.
Since small-molecules can be potentially used to treat pre- and post-exposure BoNT intoxication, research efforts to identify these antagonists have dramatically increased in recent years. However, the discovery and development of BoNT serotype A (BoNT/A) small-molecule inhibitors have long challenged researchers. Part of the difficulty in this endeavor can be attributed to the unusually large peptide substrate-enzyme interface (8) that requires a small-molecule with high affinity to effectively block substrate binding (47). Moreover, the BoNT toxin and its domains show considerable conformational flexibility, making design of effective inhibitors complicated. Despite these challenges, a number of papers have been published on the initial steps to discover and develop inhibitors of BoNT/A protease activity using different approaches. Using high throughput screening of the NCI Diversity Set, as well as a series of 4-aminoquinolines, Burnett et al. (11) identified several small-molecule inhibitors of BoNT/A, from which a common pharmacophore was predicted using molecular modeling (9). Similarly, a high throughput screen of a library of hydroxamates (6) resulted in the selection of 4-dichlorocinnamic hydroxamate as a lead structure for further development (5). Capkova et al. (12) structurally modified 2,4-dichlorocinnamic acid hydroxamate to improve its potency. On the other hand, a computational screen of 2.5 million compounds resulted in the identification of an inhibitor with a Ki of 12 μM (32), but this value was later invalidated (47). Computer-aided optimization of this inhibitor resulted in an analog that showed a two-fold improvement in inhibitory potency and displayed competitive kinetics by chelating the active site zinc atom (47).
Though the above approaches have resulted in the identification of a number of small-molecule BoNT/A inhibitors, no compound has yet advanced to pre-clinical development. The majority of these leads have only been demonstrated to be effective in enzymatic assays (11, 12, 29, 32, 47). Only a few small-molecules have been tested in cell-based assays (5, 9, 15) that involved mixing the compound with the toxin, and not by pre-loading the inhibitor. To date, none of the recently-identified BoNT/A inhibitors has been tested in a tissue-based system, and to date only two compounds were reported to have minimal in vivo activity (15).
Herein, are provided the identification of potent quinolinol-based BoNT/A small-molecule inhibitors using an integrated strategy that combined in silico screening and successive biochemical tests, including enzymatic (HPLC-based), cell-based, and tissue-based assays.